Because the xenograft cancer stem cell assays are used by all of the projects, the animal core is central to the success of this project. This assay measures self-renewal and lineage capacity, which are the hallmarks of any stem cell, therefore, as with normal stem cells, assays for cancer stem cell activity need to be evaluated for their potential to show both self-renewal and tumor propagation. The gold standard assay that fulfills these criteria is serial transplantation in animal models, which, although imperfect, is regarded as the best functional assay for these two critical criteria. In transplantation assays, cells are xenografted into an orthotopic site of immunocompromised mice that are assayed at various time points for tumor formation. To show self-renewal, cells then must be isolated from the tumors and grafted into a second recipient animal. Issues complicating transplantation assays include potential effects of the grafting site. It is known that normal stem cells can be highly dependent on signals from the surrounding stroma for function, and it is not clear what the effect may be on separating cancer stem cells from any supporting cells during the course of the assay. Experiments using mixed populations of normal and breast tumor cells in mice have shown that combining tumor cells with normal fibroblasts increases latency and decreases tumor take, whereas combining them with carcinoma-associated fibroblasts has the opposite effect. Conversely, nontumor cells placed next to tumor stroma can become independently tumorigenic, possibly due to stroma-induced genetic or epigenetic instability. The number of cells needed to form a tumor can also be affected by the addition of irradiated feeder cells or the use of Matrigel; for feeder cells, by orders of magnitude.